The transport of molecules across cell membranes is an important component of the physiologic mechanisms that mediate homeostasis at the levels of the cell and the organism as a whole. Molecules that are used directly or indirectly in the assembly of cellular components are transported from the extracellular fluid into the cell, usually by the action of specific cell-surface receptors which bind to the selected substance and mediate its uptake into specific cell types. Many hormones, enzymes, and drugs which influence cellular activity are also transported into cells by specific cell-surface receptors. Furthermore, toxic molecules which are either produced in the body (i.e. through normal or defective metabolic pathways) or introduced by ingestion or exposure can be taken up and sequestered or metabolized by certain cells. Removal of substances, both endogenously-produced and foreign substances, from extracellular fluids, such as blood or lymph, is often physiologically appropriate. However, in many instances, removal is impaired or occurs to a lesser extent than desirable and disease occurs. An example concerns LDL cholesterol, a naturally-occurring substance which must be removed at a controlled rate if abnormally elevated levels and the accompanying adverse effects are to be avoided. Hypercholesterolemia in humans is a condition characterized by elevated levels of total serum cholesterol. It is usually caused by an excess of low density lipoprotein (LDL) cholesterol or a deficiency of high density lipoprotein (HDL) cholesterol and often leads to atherosclerosis and coronary artery disease. LDL is continually formed in the blood from apolipoproteins produced by the liver. In order to maintain a steady-state level, LDL is removed from the blood at a rate equal to its formation. If LDL removal is impaired, the blood level of LDL increases and atherosclerosis is a greater risk. Atherosclerosis is by far the leading cause of death in the United States, accounting for over one-half of all deaths. (Harrison's Principles of Internal Medicine, Ed. J. D. Wilson et al., 12th ed., p. 995, McGraw-Hill, New York, 1991).
LDL particles carry approximately 60-70% of total serum cholesterol. LDL is a large spherical particle with an oily core composed of approximately 1500 cholesterol molecules, each of which is linked to a long-chain fatty acid by an ester linkage. Surrounding the core is a layer of phospholipid and unesterified cholesterol molecules, arranged in such a manner that the hydrophilic heads of the phospholipids are on the outside, and thus making it possible for the LDL to be dissolved in blood or intercellular fluid. Each LDL particle contains one molecule of Apolipoprotein B-100 (ApoB-100), a large protein molecule which is embedded in the hydrophilic coat of LDL. ApoB-100 is recognized and bound by the LDL receptor, which is present on the surfaces of cells. LDL bound to a LDL receptor is carried into the cell, in which the two are separated. The LDL receptor is recycled to the cell surface and the LDL is delivered to a lysosome. In the lysosome, LDL is processed to liberate unesterified cholesterol. The liberated cholesterol is incorporated into newly synthesized cellular membranes in all cells and, in specialized cells, is used for other purposes (e.g., steroid hormone synthesis, bile acid production).
The steady-state level of serum LDL is determined to a large extent by the number of functional hepatic LDL receptors (LDLRs), which play a central role in the removal of circulating LDL. (Brown, M. S. and Goldstein, J. L., Science, 232:34-47 (1986)) Individuals with familial hypercholesterolemia (FH) may be either heterozygous or homozygous for mutations leading to defective LDLRs and, as a result, these individuals have excess serum LDL. Other individuals who have elevated serum LDL levels may carry leaky or previously uncharacterized LDLR mutations or might be producing too much LDL due to elevated intake of dietary fat. Both FH and non-FH patients have elevated cardiovascular risk and could benefit from a therapy based on increasing the catabolism of LDL as a result of increased cellular uptake.
Thus, there exists a need to develop methods for increasing the uptake of selected substances into cells. These substances may be destined for catabolism as discussed above, or they may be designed to influence intracellular processes and thus be considered regulatory agents. Thus, cellular activity may be altered by introducing new regulatory agents which can alter specific intracellular processes into recipient cells. For example, cellular patterns of protein phosphorylation, expression of specific cellular genes, and cell growth properties may be altered by introduction of an appropriate regulatory agent into a cell. These regulatory agents may be proteins which have enzymatic activity or they may be proteins that bind specific cellular targets, targets which may be comprised of nucleic acid, protein, carbohydrate, lipid, or glycolipid.